Calcareous Spherules Produced by Intracellular Symbiotic Bacteria Protect the Sponge Hemimycale Columella from Predation Better Than Secondary Metabolites

Vol. 523: 81–92, 2015 MARINE ECOLOGY PROGRESS SERIES Published March 16 doi: 10.3354/meps11196 Mar Ecol Prog Ser FREEREE ACCESSCCESS Calcareous spherules produced by intracellular symbiotic bacteria protect the sponge Hemimycale columella from predation better than secondary metabolites Leire Garate, Andrea Blanquer, María-J. Uriz* Centre d’Estudis Avançats de Blanes (CEAB-CSIC), Access Cala Sant Francesc 14, 17300 Blanes (Girona), Spain ABSTRACT: Benthic sessile organisms in general, and sponges in particular, have developed an array of defense mechanisms to survive in crowded, resource and/or space-limited environments. Indeed, various defense mechanisms may converge in sponges to accomplish a defensive function in an additive or synergetic way, or to operate at different times during the sponge’s life cycle. Moreover, sponges harbor highly diverse microbial communities that contribute in several ways to the host’s success. Although some symbiotic bacteria produce chemical compounds that protect the sponge from predation, the possible deterrent function exerted by the calcareous coat of a sponge’s endosymbiotic bacterium has not, to date, been explored. Hemimycale columella is an Atlanto-Mediterranean sponge, which produces bioactive metabolites and has been reported to host an intracellular bacterium with a calcite envelope. Calcibacteria accumulate in high densities at the sponge periphery, forming a kind of sub-ectosomal cortex. They have been suggested to provide the sponge with several benefits, one of which is protection from predators. In this study, we assess the relative contribution of the endosymbiotic calcibacteria and bioactive compounds produced by H. columella to defend the sponge against sympatric predators. Deterrence experiments have revealed that the sponge combines >1 defense mechanism to dissuade a large array of potential predators; this represents an example of the evolutionary fixation of redundant mechanisms of defense. The chemicals deterred Paracentrotus lividus, Chromis chromis, Oblada mela- nura, and Diplodus vulgaris, but not Parablennius incognitus and Coris julis, while the spherules of the symbiotic calcibacteria significantly deterred all predators assayed. KEY WORDS: Chemical defenses · Calcifying bacteria · Sponge endosymbiosis · Sponge deterrence · Calcite spherules · Hemimycale columella · Atlanto-Mediterranean Resale or republication not permitted without written consent of the publisher INTRODUCTION Benthic sessile organisms in general, and sponges in particular, have developed an array of defense Species coexistence, which determines the biodi- mechanisms to survive in crowded, resource and/or versity of a given ecosystem, is the result of several space-limited environments. Structural materials, such long discussed, biological, and ecological mechanisms as external and internal skeletons, dermal spines, or such as environmental variation (Chesson & Warner protruding spicules serve as defense for benthic in - 1981), resource and/or niche partitioning (Chesson vertebrates and fish by protecting their soft tissues 2000), and species-specific interactions, which involve and, thus, dissuading most potential benthic preda- species-specific mechanisms of defense (Buss 1976). tors. Conversely, bioactive chemicals usually act in a *Corresponding author: [email protected] © Inter-Research 2015 · www.int-res.com 82 Mar Ecol Prog Ser 523: 81–92, 2015 less generalist way. Some chemicals may deter one or Freeman 2012). For instance, some sponge symbiotic more species from predation on the producer organ- bacteria produce chemical compounds that protect ism while they may not deter others (Becerro et al. the sponge from predation (Thacker et al. 1998, 2003). On the whole, chemical defenses have been Haber et al. 2011, Esteves et al. 2013). However, reported to significantly contribute to the structure the possible deterrent function of an endosymbiotic of sponge assemblages on coral reefs (Loh & Pawlik bacterium, other than that mediated by bioactive 2014) and have been proposed to favor complex inter- chemicals, has not been explored to date. action networks, which are responsible for in creasing Hemimycale columella (Bowerbank, 1874) is a com- species coexistence and thus biodiversity (Buss 1976, mon encrusting demosponge (Order Poecilo sclerida) Loh & Pawlik 2014). On the other hand, mineral skele- widespread in the Mediterranean and North Atlantic tons would only improve species persistence by offering sublittorals. The species, which has a reduced ecto - general protection to the organisms (Uriz et al. 2003). somal skeleton (Van Soest 2002), produces chemical Sponges are a notable component of the marine compounds with cytotoxic and antimitotic activities benthos, where they share habitat with an array of (Uriz et al. 1992, Becerro et al. 1997a) that might deter potential predators (McClintock et al. 1994, Wulff its potential predators. However, H. columella has also 2000, Santos et al. 2002, Leon & Bjorndal 2002, been reported to host an intracellular bacterium with Knowlton & Highsmith 2005). Thus, besides compet- calcifying abilities (Uriz et al. 2012). Bacteria that are ing for growth space with other benthic organisms surrounded by a 100 nm thick calcite envelope have such as algae, corals, ascidians, and bryozoans, been detected by catalyzed reporter deposition fluo- sponges must also handle predation. Indeed, sponge rescence in situ hybridization (CARD-FISH) and trans- survival has required the development of several mission electron microscopy (TEM) in high numbers defense mechanisms, which comprise the production within a particular sponge cell type called calcibac - of chemical compounds and structural elements (Uriz teriocyte (Uriz et al. 2012). Thousands of bacterium- et al. 2003, Jones et al. 2005), along with cryptic produced calcite spherules are accumulated at the growth habits (Bertolino et al. 2013). sponge periphery, forming a kind of sub-ectosomal Many of the bioactive compounds produced by cortex that mimics a rudimentary exoskeleton (Uriz sponges with antimitotic, cytotoxic, antibacterial, et al. 2012). It has been proposed that this unusual, and/or antitumor activities (Amade et al. 1987, Uriz intimate symbiosis, which is vertically transmitted to et al. 1992, Becerro et al. 1994, Monks et al. 2002, progeny and constantly present in all populations of H. Sipkema et al. 2005, Blunt et al. 2009, Hardoim & columella examined along the western Me diterranean Costa 2014) inhibit the settlement of foreign larvae in sublittoral zone, purportedly provides the sponge with the proximity of the sponge (Martin & Uriz 1993, several benefits, among which protection from pre - Becerro et al. 1997a, 2003, De Caralt et al. 2013) or dators has been highlighted (Uriz et al. 2012). deter predation (Uriz et al. 1996, Ribeiro et al. 2010, In this study, we aimed to assess the relative contri- Arias et al. 2011). Spicules have also been reported to butions of the calcite spherules of endosymbiotic cal- dissuade sponge predators to some extent (Burns & cibacteria and the bioactive compounds produced by Ilan 2003, Hill et al. 2005, Jones et al. 2005, but see H. columella to sponge defense, and whether the Chanas & Pawlik 1995). Thus, various defense mech- combination of secondary metabolites and the cal- anisms converge in most sponges to accomplish a cibacterial calcareous envelope exerted a synergetic defensive function in an additive or synergetic way, effect in deterring potential predators from feeding or to operate at different times during the sponge’s on the sponge. With this aim, we conducted several life cycle (Uriz et al. 1996). However, the opposite is deterrence experiments, both in the laboratory and also true: multiple functions have also been reported in the sponge’s habitat, with an array of sympatric for a sole defense mechanism (Thacker et al. 1998, potential predators (echinoderms and fishes). Becerro et al. 1997a). Furthermore, the efficiency of a deterrent mechanism can vary according to the predator (Becerro et al. 2003), which makes the MATERIALS AND METHODS results of deterrence assays difficult to generalize. To add to the complexity of defense mechanisms, Sampling, sponge identification, and location of sponges harbor highly diverse microbial communi- bacteria ties (e.g. Blanquer et al. 2013), which form stable symbiotic associations and contribute in several ways Between 8 and 12 individuals of Hemimycale col- to the host’s success (Taylor et al. 2007, Thacker & umella were randomly collected from the Blanes lit- Garate et al.: Chemical defenses versus endosymbiotic calcibacteria in sponge deterrence 83 toral zone, NW Mediterranean (41°40.12’N, 2°47.10’E), were initiated. Once the experiments were com- in each of 3 sampling dives, to prepare the artificial pleted all individuals were taken back to their natu- food used in the experiments. The species was taxo- ral habitat. nomically identified by phenotypic characters, such as external morphology (i.e. thick encrusting shape, presence of characteristic rounded pore sieves with Chemical extraction elevated rims, pale orange to pink color, and spicule shape [strongyles to styles], size [320 to 461 μm × 2.5 Ca. 25 g of fresh sponges, corresponding to 40 ml in to 7.5 μm), and plumose arrangement [Van Soest volume (according to the water volume displaced 2002]). The sponge samples were taken to the labo- when submerged in a measuring cylinder), were ratory in hermetic, seawater-filled bowls, blended, freeze-dried

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